一种凋亡相关蚯蚓丝氨酸蛋白酶的纯化、活性鉴定及部分性质研究

通过多级柱层析,从赤子爱胜蚓抽提物（一组抗肿瘤活性蛋白成分）中纯化得到凋亡相关丝氨酸蛋白酶1（apoptosis-related serine protease 1, ARSP1）,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测得其表观分子质量为28 ku.ARSP1非变性PAGE图谱为相连的多条带,质谱图为多头峰,MALDI-TOF-MS测得各主峰相对分子质量为24 645,25 052和25 281,等电聚焦电泳测得等电点pI＜3.8.测得ARSP1 N端25个氨基酸序列为：I(V)IGGT(S)N(D)ASPGEFPWQLSQTRGGSHS,N端序列比较结果显示其与丝氨酸蛋白酶类高度同源.体外实验中,不仅通过凋亡细胞的相差显微观察验证了ARSP1的细胞杀伤活性,而且进一步通过荧光抗体技术对其直接杀伤细胞活性进行了定位研究.Schiff's试剂糖蛋白染色法鉴定ARSP1为糖蛋白（或糖肽）,纤维蛋白平板法测得ARSP1同时具有纤溶酶和纤溶酶原激活酶活性,进一步通过苯甲磺酰氟(PMSF)对其纤溶酶活性的抑制实验,证明属于丝氨酸蛋白酶类.     

Chemotypes of Fusobacterium nucleatum lipopolysaccharides

Lipopolysaccharides (LPS) were isolated from 20 strains of Fusobacterium nucleatum and examined by paper chromatography, gas liquid chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-dexoxy-octonate (KDO). The LPS had in common glucosamine, L-glycero-D-manno-heptose, glucose and KDO. The KDO content was low. Galatose, rhamnose and D-glycero-D-manno-heptose were found in some strains. Based on the sugar composition of the LPS, the F. nucleatum strains could be classified into six chemotypes.     

Characterization of aid1, a Novel Gene Involved in Fusobacterium nucleatum Interspecies Interactions

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.     

Fusobacterium nucleatum subsp. fusiforme subsp. nov. and Fusobacterium nucleatum subsp. animalis subsp. nov. as additional subspecies within Fusobacterium nucleatum.

Using a variety of physiological, biochemical, and molecular systematic analyses, we have shown previously that there are four groups within the species Fusobacterium nucleatum. Two of these groups of strains correspond to the recently proposed taxa F. nucleatum subsp. nucleatum and F. nucleatum subsp. polymorphum. In this paper we show that the two remaining groups are distinct and formally propose that they should be recognized as F. nucleatum subsp. fusiforme (type strain, NCTC 11326) and F. nucleatum subsp. animalis (type strain, NCTC 12276). The tests which we used did not allow a full assessment of the status of F. nucleatum subsp. vincentii compared with F. nucleatum subsp. nucleatum.     

Heterogeneity within Fusobacterium nucleatum, proposal of four subspecies

Fusobacterium nucleatum strains, isolated from man and animals, were shown to comprise four centres of variation within the species by using a variety of biochemical tests. DNA-DNA hybridization data indicated that they should differences between the groups to warrant their placement into four subspecies for which we propose the following: F. nucleatum subsp. nucleatum (commonly isolated from diseased sites), F. nucleatum subsp. polymorphum (from healthy sites, most frequently isolated), F. nucleatum subsp. fusiforme (from healthy sites, most frequently isolated), F. nucleatum subsp. fusiforme (from healthy sites, rarely isolated) and F. nucleatum subsp. animalis from the colon of animals.     

Haemagglutination and haemolysis by oral Fusobacterium nucleatum

Haemagglutination and haemolytic activity of 80 Fusobacterium nucleatum isolates from human and animal origin, on different human blood types was evaluated. All the isolates were able to agglutinate erythrocytes and the most were either alpha-haemolytic or beta-haemolytic. No specificity between haemolysin or haemagglutinin and blood type was observed. Haemagglutination activity was inhibited when D-galactose, D-lactose or D-raffinose were used. Haemagglutination and haemolysis may be important factors in the pathogenesis of human and animal periodontal diseases.     

Characterization of the Novel Fusobacterium nucleatum Plasmid pKH9 and Evidence of an Addiction System

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

Functional and Structural Characterization of Vibrio cholerae Extracellular Serine Protease B,VesB

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser. Using synthetic peptides, VesB efficiently cleaved a trypsin substrate, but not chymotrypsin and elastase substrates. The reversible serine protease inhibitor, benzamidine, inhibited VesB and served as an immobilized ligand for VesB affinity purification, further indicating its relationship with trypsin-like enzymes. Consistent with this family of serine proteases, N-terminal sequencing implied that the propeptide is removed in the secreted form of VesB. Separate mutagenesis of the activation site and catalytic serine rendered VesB inactive, confirming the importance of these features for activity, but not for secretion. Similar to trypsin but, in contrast to thrombin and other coagulation factors, Na⁺ did not stimulate the activity of VesB, despite containing the Tyr²⁵⁰ signature. The crystal structure of catalytically inactive pro-VesB revealed that the protease domain is structurally similar to trypsinogen. The C-terminal domain of VesB was found to adopt an immunoglobulin (Ig)-fold that is structurally homologous to Ig-folds of other extracellular Vibrio proteins. Possible roles of the Ig-fold domain in stability, substrate specificity, cell surface association, and type II secretion of VesB, the first bacterial multidomain trypsin-like protease with known structure, are discussed.     

Characterization of the novel Fusobacterium nucleatum plasmid pKH9 and evidence of an addiction system

Fusobacterium nucleatum is an important oral anaerobic pathogen involved in periodontal and systemic infections. Studies of the molecular mechanisms involved in fusobacterial virulence and adhesion have been limited by lack of systems for efficient genetic manipulation. Plasmids were isolated from eight strains of F. nucleatum. The smallest plasmid, pKH9 (4,975 bp), was characterized and used to create new vectors for fusobacterial genetic manipulation. DNA sequence analysis of pKH9 revealed an open reading frame (ORF) encoding a putative autonomous rolling circle replication protein (Rep), an ORF predicted to encode a protein homologous to members of the FtsK/SpoIIIE cell division-DNA segregation protein family, and an operon encoding a putative toxin-antitoxin plasmid addiction system (txf-axf). Deletion analysis localized the pKH9 replication region in a 0.96-kbp fragment. The pKH9 rep gene is not present in this fragment, suggesting that pKH9 can replicate in fusobacteria independently of the Rep protein. A pKH9-based, compact Escherichia coli-F. nucleatum shuttle plasmid was constructed and found to be compatible with a previously described pFN1-based fusobacterial shuttle plasmid. Deletion of the pKH9 putative addiction system (txf-axf) reduced plasmid stability in fusobacteria, indicating its addiction properties and suggesting it to be the first plasmid addiction system described for fusobacteria. pKH9, its genetic elements, and its shuttle plasmid derivatives can serve as useful tools for investigating fusobacterial properties important in biofilm ecology and pathogenesis.     

Fusobacterium nucleatum in periodontal health and disease

The pathogenesis of periodontitis involves the interplay of microbiota present in the subgingival plaque and the host responses. Inflammation and destruction of periodontal tissues are considered to result from the response of a susceptible host to a microbial biofilm containing gram-negative pathogens. Antimicrobial peptides are important contributors to maintaining the balance between health and disease in this complex environment. These include several salivary antimicrobial peptides such as β-defensins expressed in the epithelium and LL-37 expressed in both epithelium and neutrophils. Among gram-negative bacteria implicated in periodontal diseases, Fusobacterium nucleatum, is one of the most interesting. This review will focus on expression, function, regulation and functional efficacy of antimicrobial peptides against F. nucleatum. We are looking for how the presence of F. nucleatum induces secretion of peptides which have an impact on host cells and modulate immune response.     

Extraction and Characterization of the Smooth-Type Lipopolysaccharide from Fusobacterium nucleatum JCM 8532 and Its Biological Activities

The lipopolysaccharide (LPS) of Fusobacterium nucleatum JCM 8532 was isolated by hot-phenol water extraction. Most of the LPS was extracted in the phenolic phase and shown to be the smooth-type, whereas the aqueous phase contained mainly rough-type LPS. The chemical composition of the LPS was similar to that reported in other studies, but D -quinovosamine, which may be a major component of O-antigenic polysaccharide, and 3-deoxy-D -manno-2-octulosonic acid (Kdo) were detected for the first time by gas chromatography-mass spectrometry. The biological activities of smooth-type LPS, including limulus activity, lethal toxicity, pyrogenicity, and B lymphocyte mitogenicity, were comparable to those of enterobacterial LPS. Smooth-type LPS inhibited the cell growth and DNA synthesis of adult and fetal human gingival fibroblasts in a dose-dependent manner, suggesting that LPS may play a role in the occurrence of human gingivitis.     

Isolation, purification and characterisation of 2-oxoglutarate reductase from Fusobacterium nucleatum

2-Oxoglutarate reductase from Fusobacterium nucleatum was isolated by thiol-disulphide interchange covalent chromatography. The enzyme was purified approximately 4000-fold and had a molecular mass of 68 kDa. The Michaelis constants for 2-oxoglutarate and NADH were 6.4 x 10(-5) and 0.4 x 10(-5), respectively. The involvement of sulphahydryl groups in catalysis was shown from the inhibition of 2-oxoglutarate reduction in the presence of 2,2'-dipyridyl disulphide and reactivation with 2-mercaptoethanol. Allosteric effectors did not alter the rate of the reaction, or the enzyme stability. With the exception of 2-oxoglutarate, none of the other oxo-acids such as oxaloacetate, pyruvate, 2-oxobutyrate and glyoxylate were reduced. Although 2-oxoglutarate oxidised NADPH to a limited extent (3%), the enzyme was almost entirely specific towards NADH. 2-Oxoglutarate reductase was stable at 45 degrees C for 10 min, while incubation at 60 degrees C abolished all activity.     

Phenotypic and genotypic analyses of clinical Fusobacterium nucleatum and Fusobacterium periodonticum isolates from the human gut

Fusobacterium nucleatum is a Gram-negative anaerobic rod that is part of the normal human microflora, and has also been associated with various infections. Bacterial strains belonging to the species are typically heterogeneous in both phenotype and genotype, which can hinder their identification in a clinical setting. The majority of F. nucleatum isolates originate from oral sites, however the species is also a resident of the human gastrointestinal tract. The aim of this study was to compare F. nucleatum isolates from human intestinal biopsy samples to try and determine whether isolates from this site are divergent from oral isolates. We used a variety of phenotypic and genotypic markers to compare 21 F. nucleatum and Fusobacterium periodonticum isolates from the GI tract to oral isolates and recognized type strains in order to study heterogeneity within this set. 16S rDNA and rpoB gene sequence analysis allowed us to build phylogenetic trees that consistently placed isolates into distinct clusters. 16S rDNA copy number analyses using Denaturing Gradient Gel Electrophoresis (DGGE) demonstrated potential for use as a method to examine clonality amongst species. Phenotypic analyses gave variable results that were generally unhelpful in distinguishing between phylogenetic clusters. Our results suggest that a) F. periodonticum isolates are not restricted to the oral niche; b) phenotypic classification is not sufficient to subspeciate isolates; c) heterogeneity within the species is extensive but constrained; and d) F. nucleatum isolates from the gut tend to identify with the animalis subspecies.     

Pathway of lysine degradation in Fusobacterium nucleatum.

Lysine was fermented by Fusobacterium nucleatum ATCC 25586 with the formation of about 1 mol each of acetate and butyrate. By the use of [1-14C]lysine or [6-14C]lysine, acetate and butyrate were shown to be derived from both ends of lysine, with acetate being formed preferentially from carbon atoms 1 and 2 and butyrate being formed preferentially from carbon atoms 3 to 6. This indicates that the lysine carbon chain is cleaved between both carbon atoms 2 and 3 and carbon atoms 4 and 5, with the former predominating [1-14C]acetate was also extensively incorporated into butyrate, preferentially into carbon atoms 3 and 4. Cell-free extracts of F. nucleatum were shown to catalyze the reactions of the 3-keto,5-aminohexanoate pathway of lysine degradation, previously described in lysine-fermenting clostridia. The 3-keto,5-aminohexanoate cleavage enzyme was partially purified and shown to have properties much like those of the clostridial enzyme. We conclude that both the pathway and the enzymes of lysine degradation are similar in F. nucleatum and lysine-fermenting clostridia.     

Cloning and characterization of the L-cysteine desulfhydrase gene of Fusobacterium nucleatum

Pseudomonas stutzeri, in addition to being transformed by duplex DNA, is also transformed by the sense or antisense strand of the genetic marker employed (hisX(+)) or by heat-denatured chromosomal DNA. Transformation was absent in non-competent cells and in mutants defective for pilus biogenesis (pilA, pilC) and function (pilT) or DNA translocation into the cytoplasm (comA). Uptake of (3)H-thymidine-labeled single-stranded DNA was hardly detectable reflecting the 20- to 60-fold lower transformation compared to duplex DNA. The results suggest that the steps in natural transformation also accommodate single-stranded DNA and that DNA translocation from the periplasm into the cytoplasm is not necessarily coupled to the degradation of a complementary strand. Small DNA single-stranded fragments are thus not excluded from horizontal gene transfer by transformation.     

Identification,Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.Protein secretion plays important roles in bacterial life, as many of the secreted proteins are involved in biological processes that are fundamental to the survival and environmental adaptations of the cells. Gram-negative bacteria have evolved a number of secretion systems that utilize different secretion apparatus and mechanisms (9). The classical autotransporter secretion pathway belongs to the type V secretion system (8, 12, 13). Compared to other types of secretion mechanisms, the autotransporter system is unique in that all the components that are required for protein translocation are contained within a single polypeptide. Structurally, autotransporters are characterized by three domains: (i) an N-terminal signal sequence that is recognized by the Sec translocon; (ii) a central passenger domain (or α-domain) that contains the effector molecule and is highly variable; and (iii) a C-terminal translocation domain (or β-/autotransporter domain) that is conserved in length (250 to 300 amino acids) but varies in primary structure (7, 12, 55). In most cases, the β-domain contains 12 antiparallel strands of 9 to 12 residues that, upon integration into the outer membrane, form a β-barrel conformation (27, 57). Another conserved feature of the β-domain is the presence at the C terminus of a sequence motif, (Y/V/I/F/W)-X-(F/W), that is characterized by alternating hydrophobic and hydrophilic residues and ends with either a tryptophan or a phenylalanine (12, 16, 27). The integrity of this end motif seems to be required for protein translocation, as deletion of certain residues in this sequence impairs protein secretion. The secretion process of the autotransporter is initiated by the signal sequence, which directs the translocation of the protein precursor across the inner membrane into the periplasmic space via the Sec system. Once inside the periplasm, the β-domain inserts into the outer membrane and adopts the structure of a β-barrel through which the passenger domain is translocated to the cell surface, where it may exist as a membrane-anchored protein covalently linked to the β-domain or be cleaved from the β-domain as a result of proteolysis (7).Since the discovery of the gonococcal immunoglobulin A1 protease (35), the first autotransporter, and especially with the advent of genome sequencing technology, autotransporters have been identified in many bacterial species (18, 19, 28, 33). Functions assigned to autotransporters are mostly associated with bacterial pathogenicity, which includes adhesion and invasion into host cells, biofilm formation, and cytotoxicity (11, 54). In the present study, we identified and analyzed an autotransporter, PfaI, from a pathogenic Pseudomonas fluorescens strain isolated from diseased fish. We found that, like many of the autotransporters identified in other pathogens, PfaI is a virulence factor that is involved in interactions with host cells and modulation of host immune responses via a protease effector. In addition, we found that the autotransporter property of PfaI could be exploited for the delivery and surface display of an immunoprotective antigen.     

Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis

AIMS: To develop a semi-quantitative method for evaluating co-aggregation reactions among three bacterial species, and to examine the influence of Fusobacterium nucleatum on the adherence of Porphyromonas gingivalis. METHODS AND RESULTS: The method involves coating hydroxyapatite (HAP) discs with streptococcal cells and treatment with radio-labelled bacterial cell suspensions. The sensitivity of the method was estimated by comparison with a turbidometric co-aggregation assay. Results from the two methods were in close agreement. Streptococcus mitis-coated HAP discs were immersed in a 3H-labelled Fus. nucleatum cell suspension and then a 14C-labelled P. gingivalis cell suspension. The discs were then pyrolysed to recover and quantify the released 3H and 14C radioactivity. The number of Fus. nucleatum cells on the discs increased with immersion time and this, in turn, resulted in elevated adherence of P. gingivalis. CONCLUSION: The data indicate that the method closely reflects co-aggregation characters, and that Fus. nucleatum has a positive effect on the adherence of P. gingivalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present method, which is designed to mimic the oral environment, should prove useful in the semi-quantitative evaluation of co-aggregation reactions.     

Sizing the Fusobacterium nucleatum genome by pulsed-field gel electrophoresis

Abstract The β-galactosidase (β-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli . Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum . Its sequence is very similar to a Leuconostoc lactis β-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis , was four-fold higher in cells grown in lactose compared to those grown in glucose. The presence of the β-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.